Rojas-Henríquez Victor, Olate-Briones Alexandra, Salazar Celia, Escobedo Noelia, Herrada Andrés A
Lymphatic Vasculature and Inflammation Research Laboratory, Facultad de Ciencias de la Salud, Instituto de Ciencias Biomédicas, Universidad Autónoma de Chile, Talca, Chile.
Curr Protoc. 2025 Jun;5(6):e70160. doi: 10.1002/cpz1.70160.
The lymphatic vasculature (LV) plays a crucial role in regulating the trafficking of immune cells into lymph nodes during homeostasis and under inflammatory conditions. LV dysfunction has been linked to various inflammatory and neuroinflammatory diseases. Thus, assessing the LV coverage area using confocal immunofluorescence microscopy is essential for accurately determining the extent of inflammation in the tissue. However, manual selection processes may require considerable time to finalize and are inherently more prone to errors, which can compromise the consistency of results among various users and lead to imprecise measurements. Here, we have developed an improved protocol to determine the area of LV covering the intestinal and meningeal tissues. By using this protocol, regions of interest can be identified more accurately and efficiently than through manual measurement, allowing for efficient analysis of changes in LV coverage during inflammation. Colon tissue and meninges obtained from mice under different experimental conditions were stained with LV-specific antibodies. Confocal microscopy was used to capture all images. Here, the manual selection protocol was compared with our semi-automatic selection protocol using the Fiji software Threshold tool. Using this tool, we successfully automated the selection of regions of interest, achieving greater accuracy in our values and minimizing both errors and the time spent by the operator. Additionally, this tool produces much more specific selections than those performed manually, and the results are more accurate. Finally, it is essential to note that this protocol can be adapted for use with other microscopy techniques, provided the image has sufficient contrast against the background and the scale size is known. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Immunofluorescence of Colon Tissue Section and Whole-Mount Meninges Basic Protocol 2: Semi-Automated Analysis of Lymphatic Vasculature Using the Fiji Software.
淋巴脉管系统（LV）在稳态和炎症条件下调节免疫细胞向淋巴结的运输中起着关键作用。LV功能障碍与多种炎症和神经炎症性疾病有关。因此，使用共聚焦免疫荧光显微镜评估LV覆盖面积对于准确确定组织中的炎症程度至关重要。然而，手动选择过程可能需要相当长的时间才能完成，而且本身更容易出错，这可能会影响不同用户之间结果的一致性，并导致测量不准确。在这里，我们开发了一种改进的方案来确定LV覆盖肠道和脑膜组织的面积。通过使用该方案，可以比手动测量更准确、高效地识别感兴趣区域，从而能够有效分析炎症期间LV覆盖面积的变化。从处于不同实验条件下的小鼠获取的结肠组织和脑膜用LV特异性抗体进行染色。使用共聚焦显微镜拍摄所有图像。在这里，使用Fiji软件的阈值工具将手动选择方案与我们的半自动选择方案进行了比较。使用该工具，我们成功实现了感兴趣区域选择的自动化，在数值上实现了更高的准确性，同时将误差和操作员花费的时间降至最低。此外，该工具产生的选择比手动操作更具特异性，结果也更准确。最后，必须注意的是，只要图像与背景有足够的对比度且已知比例尺大小，该方案可适用于其他显微镜技术。© 2025威利期刊有限责任公司。基本方案1：结肠组织切片和全层脑膜的免疫荧光 基本方案2：使用Fiji软件对淋巴脉管系统进行半自动分析
